עבודה זו נתמכה המענק SCCOR &quot;מחלת AAA: מנגנונים, ריבוד, וטיפול&quot; (HL083800 P50-03) מן המכון הלאומי לבריאות

<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עכברים מורדמים עם בשאיפה isoflurane 2.0-3.0ml / ל &#39; </li><li style=";text-align:right;direction:rtl"> הבטן היא מגולח מצוחצח עם אלכוהול בבטאדין. </li><li style=";text-align:right;direction:rtl"> העכבר מונח פרקדן על שדה הניתוח. </li><li style=";text-align:right;direction:rtl"> כפפות סטריליות שחוקים על ידי המנתח, ואת הכלים המשמשים כבר מעוקר עם מעקר חרוז חם. </li><li style=";text-align:right;direction:rtl"> חתך ארוך בבטן קו האמצע הוא עשה. תכולת הבטן הם חזר בו מחוץ הבטן עם גזה סטרילית לחשוף את אבי העורקים בבטן. </li><li style=";text-align:right;direction:rtl"> אבי העורקים בבטן מבודד ברמה של עורק הכליה משמאל הסתעפות. </li><li style=";text-align:right;direction:rtl"> הסניפים של אבי העורקים בבטן חשופים ligated עם 10-0 התפרים. סניפים כל אבי העורקים בתוך 1.0cm של הסתעפות הם ligated. </li><li style=";text-align:right;direction:rtl"> זמניים 6.0 חיבורי אותיות משי ממוקמים סביב חלקים הפרוקסימלית ומ הדיסטלי של האאורטה </li><li style=";text-align:right;direction:rtl"> Aortotomy נוצר ב הסתעפות עם מחט 30-מד. </li><li style=";text-align:right;direction:rtl"> חום קוני צינורות פוליאתילן (PE-10) הוא הציג דרך aortotomy ומאובטחת עם עניבה. </li><li style=";text-align:right;direction:rtl"> בעזרת שקית מלח תלוי בגובה של 136 ס&quot;מ לכייל 100 מ&quot;מ כספית, אבי העורקים מתמלא מלוחים המכיל 4.5 U / mL אני סוג elastase חזירי הלבלב. אבי העורקים, בדרך כלל, מתרחב אל% 150-170 בקוטר המקורי במהלך זלוף elastase 5-15 דקות. </li><li style=";text-align:right;direction:rtl"> קטטר זלוף מוסר אז aortotomy נסגר עם תפר 10-0 כדי למנוע התכווצות. </li><li style=";text-align:right;direction:rtl"> לאחר aortotomy סגור, המיתר דיסטלי מוסר. </li><li style=";text-align:right;direction:rtl"> אם יש דימום מועט או ללא מ aortotomy המתוקן, ליגטורה הפרוקסימלית מוסר. אבי העורקים התרחב מתמלא בדם באופן מיידי. </li><li style=";text-align:right;direction:rtl"> המעיים הם חזרו הבטן, ואת הפצע סגור בשתי שכבות ניילון עם תפר / פוליפרופילן 6-0. </li><li style=";text-align:right;direction:rtl"> 0.25% Bupivicaime טיפות (appx טיפה אחת עבור כל סנטימטר של גודל החתך) ממוקמות על הפצע מנה אחת של Caprofen (5mg/kg) ניתנת תת עורי. </li><li style=";text-align:right;direction:rtl"> העכבר ממוקם על שטח חמים בדרך כלל מתאוששת בתוך 15 דקות. </li></ol><img src="/files/ftp_upload/1280/fig1280.png" alt="איור 1" /> באיור 1. תוצאות נציג Elastase המושרה AAA.

<ol>
	<li>Anidjar, S., Salzmann, J. L., Gentric, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastase-induced+experimental+aneurysms+in+rats.">Elastase-induced experimental aneurysms in rats.</a> Circulation. 82 (3), 973-973 (1990).</li><li>Pyo, R., Lee, J. K., Shipley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+disruption+of+matrix+metalloproteinase-9+(gelatinase+B)+suppresses+development+of+experimental+abdominal+aortic+aneurysms.">Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms.</a> J Clin Invest. 105 (11), 1641-1641 (2000).</li><li>Carsten, C. G., Calton, W. C., Johanning, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastase+is+not+sufficient+to+induce+experimental+abdominal+aortic+aneurysms.">Elastase is not sufficient to induce experimental abdominal aortic aneurysms.</a> J Vasc Surg. 33 (6), 1255-1255 (2001).</li></ol>

The authors have nothing to disclose.

מחקרים קודמים ציין וריאציה משמעותית הרבה-to-הרבה בפיתוח AAA מ שונה ההכנות elastase מסחרי 3. לכן, חשוב לעקוב אחר המון הייצור וכן יש קבוצות ביקורת מתאימות כדי לתעד את היכולת של התכשיר כדי לגרום AAAS. מעניין, הרגישות פיתוח AAA במודל זה תלוי המתח העכבר משמש 3. העכבר משמש הווידאו הנוכחי של זן C57Bl / 6 אשר יש התפתחות עקבית של AAA באמצעות פרוטוקול התווה. ריכוז Elastase ו / או זמן הדגירה יכול להיות מותאם כדי להתאים זנים אחרים. למרות הטכניקה של יצירת AAA עם elastase בעכברים הוא מאתגר מבחינה טכנית, היא טכניקה חשובה לחקר המנגנונים המולקולריים שבבסיס של היווצרות פיתוח AAA ו. עיסוק שינויים טכניים גרמו שיעור הצלחה של מעל 90% במעבדה שלנו. אנו מאמינים כי הווידאו והטקסט המלווה יסייע להפחית את עקומת הלמידה עבור מעבדות המבקשים להשתמש בטכניקה זו. פתרון בעיות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הסניפים של אבי העורקים בבטן יש ligated עם 10-0 התפרים, מניעת דימום במהלך המבצע והרחבת מספיק במהלך עירוי elastase בגלל דולף. </li><li style=";text-align:right;direction:rtl"> חשוב יש שדה ההפעלה ברורה ורחבה. לכן, להשתמש 6-0 תפר במקום מלחציים זמנית לחסום את אבי העורקים </li><li style=";text-align:right;direction:rtl"> לומן של אבי העורקים בבטן יש לפנות עם תמיסת מלח כדי למנוע קרישי דם לאחר הניתוח. </li><li style=";text-align:right;direction:rtl"> חשוב לחמם את קצה הצינור קוני PE-10 צריך להיות חלק כדי להימנע מגרד את שכבת האנדותל כאשר מוכנס. </li><li style=";text-align:right;direction:rtl"> בועות האוויר בתוך הכלים יש להימנע כאשר יציקת elastase וכן כאשר aortotomy סגור. </li></ol>

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Type I porcine pancreatic elastase</td>
<td>Reagent</td>
<td>Sigma</td>
<td>E1250</td>
<td>&nbsp;</td>
<tr>
<td>polyethylene tubing (PE-10)</td>
<td>&nbsp;</td>
<td>CLAY ADAMS</td>
<td>427400</td>
<td>&nbsp;</td>
<tr>
<td>CHANGE-A-TIP</td>
<td>&nbsp;</td>
<td>Bovie</td>
<td>18010-00 (FST)</td>
<td>cautery knife</td>
</tr>
<tr>
<td>LEICA MZ6</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>stereomicroscope</td>
</tr>
<tr>
<td>LEICA DFC290HD</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>USB camera</td>
</tr>
<tr>
<td>Image Pro Express ver. 6.0</td>
<td>&nbsp;</td>
<td>Media Cybernetics</td>
<td>&nbsp;</td>
<td>software</td>
</tr>		</tbody>
		</table>
		</div>

creation of murine experimental abdominal aortic aneurysms with elastase 

עירוי intraluminal חלוף של elastase הלבלב חזירי לתוך מגזר infrarenal של אבי העורקים בבטן הוא מודל החיה הנפוצה ביותר של מפרצת אבי העורקים הבטני (AAA) מאז זה תוארה לראשונה על ידי Anidjar ועמיתיו בחולדות. 1 הרציונל הפיתוח שלה התבססה על אופי שיבשו של ואלסטין שנצפתה AAAS. מודל זה עכברוש שונתה כדי לייצר AAAS באזור infrarenal אבי העורקים של עכברים. 2 מודל יש את היכולת להוסיף תובנה רחבה לתוך pathobiology של AAA בשל הופעתה של מהונדס רבים נוקאאוט בעכברים גנים. יתר על כן, היא פלטפורמה מעשית לבדוק סוכני טיפולית פוטנציאל AAA. בסרטון הזה, אנחנו מדגימים את הליך עירוי elastase AAA בשימוש במעבדה שלנו. עכברים מורדמים באמצעות isoflurane 2.5%, ו laparotomy מבוצעת בתנאים סטריליים. Aortais בטן מבודד בסיוע stereomicroscope ההפעלה (לייקה). לאחר הצבת חיבורי אותיות זמני סביב האאורטה הפרוקסימלית ומ דיסטלי, aortotomy נוצר ב הסתעפות עם קצה מחט 30-מד. קטע חום מחודדות של צינורות פוליאתילן PE-10 הוא הציג דרך aortotomy ומאובטחת. לומן העורקים היא perfused לאחר מכן ב 100 מ&quot;מ כספית עם סוג המכיל תמיסת מלח אני elastase חזירי הלבלב (4.5 U / mL: Sigma כימית ושות&#39;) במשך 5-15 דקות. לאחר הסרת הקטטר זלוף, aortotomy הוא תוקן ללא התכווצות של לומן.

Watch this Scientific Journal Video about Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase at JoVE.com

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase

וידאו זה מראה כיצד לגרום מפרצת אבי העורקים הבטני (AAA) בעכברים באמצעות עירוי intraluminal חולף של elastase הלבלב חזירי לתוך מגזר infrarenal של אבי העורקים בבטן. המודל יש את היכולת להוסיף תובנות לתוך pathobiology רחב של AAA בשל הופעתה של מהונדס רבים נוקאאוט בעכברים גנים.

יצירת ניסויית מפרצת אבי העורקים הבטני Murine עם Elastase

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of ...

creation-murine-experimental-abdominal-aortic-aneurysms-with

Google

Research

JoVE Journal

Biology

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase 

24.4K Views.  Stanford University School of Medicine. וידאו זה מראה כיצד לגרום מפרצת אבי העורקים הבטני (AAA) בעכברים באמצעות עירוי intraluminal חולף של elastase הלבלב חזירי לתוך מגזר infrarenal של אבי העורקים בבטן. המודל יש את היכולת להוסיף תובנות לתוך pathobiology רחב של AAA בשל הופעתה של מהונדס רבים נוקאאוט בעכברים גנים.

Video: Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase

Aortic Ring Assay

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis assays involve the study of individual endothelial cells in culture conditions (1). The aortic ring assay is an angiogenesis model that is based on organ culture. In this assay, angiogenic vessels grow from a segment of the aorta (modified from (2)). Briefly, mouse thoracic aorta is excised, the fat layer and adventitia are removed, and rings approximately 1 mm in length are prepared. Individual rings are then embedded in a small solid dome of basement matrix extract (BME), cast inside individual wells of a 48-well plate. Angiogenic factors and inhibitors of angiogenesis can be directly added to the rings, and a mixed co-culture of aortic rings and other cell types can be employed for the study of paracrine angiogenic effects. Sprouting is observed by inspection under a stereomicroscope over a period of 6-12 days. Due to the large variation caused by the irregularities in the aortic segments, experimentation in 6-plicates is strongly advised. Neovessel outgrowth is monitored throughout the experiment and imaged using phase microscopy, and supernatants are collected for measurement of relevant angiogenic and anti-angiogenic factors, cell death markers and nitrite.

Angiogenesis, the sprouting of blood vessels from pre-existing vasculature, is associated with both natural and pathological processes. Here we demonstrate an aortic ring assay that allows angiogenic potentiators and inhibitors to be directly added to aortic rings in culture. Sprouting and neovessel outgrowth can be determined by inspecting the aortic rings over a period of 6-12 days.

אבי העורקים Assay טבעת

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis ...

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

אבי העורקים צמצום רוחבי של עכברים

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

בידוד והתרבות ראשית של לתאי אנדותל עכבר אב העורקים

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

גישה חלופית עבור הכנת דוגמאות עם מספר סלולרי נמוך עבור ניתוח TEM

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Murine אבי העורקים למחוץ פגיעה: יעיל<em&gt; בויבו</em&gt; מודל של תאים שרירים חלקה תפוצת ו אנדותל פונקציה

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.

מניפולציה ממסדית וגנטית של אורגנוידים של מורין הפטוציט

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

יישום של תנועת ראש פסיבית ליצירת תאוצות מוגדרות בראשי מכרסמים

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

This paper describes an explant culture-based method for the isolation and culturing of primary, patient-specific human aortic smooth muscle cells and dermal fibroblasts. Furthermore, a novel method is presented for measuring cell contraction and subsequent analysis, which can be used to study patient-specific differences in these cells.

בידוד של תאי שריר חלק אבי העורקים העיקריים הספציפיים למטופל ומדידות כיווץ חצי-קוונטיטיביות בזמן אמת במבחנה

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in ...

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. Currently, there are several distinct experimental models of AAA, each emphasizing a different aspect in the pathogenesis of AAA. The elastase-induced AAA model is the second most used rodent AAA model. This model involves direct infusion or application of porcine pancreatic elastase (PPE) to the infrarenal segment of the aorta. Due to technical challenges, most elastase-induced AAA model nowadays is performed with the external application rather than an intraluminal infusion of PPE. The infiltration of elastase will cause degradation of elastic lamellae in the medial layers, resulting in the loss of aortic wall integrity and subsequent dilation of the abdominal aorta. However, one disadvantage of the elastase-induced AAA model is the inevitable variation of how the surgery is performed. Specifically, the surgical technique of isolating the infrarenal segment of the aorta, the material used for aorta wrapping and PPE incubation, the enzymatic activity of PPE, and the time duration of PPE application can all be important determinants that affect the eventual AAA formation rate and aneurysm diameter. Notably, the difference in these factors from different studies on AAA can lead to reproducibility issues. This article describes a detailed surgical process of the elastase-induced AAA model through direct application of PPE to the adventitia of the infrarenal abdominal aorta in the mouse. Following this procedure, a stable AAA formation rate of around 80% in male and female mice is achievable. The consistency and reproducibility of AAA studies using an elastase-induced AAA model can be significantly enhanced by establishing a standard surgical procedure.

The present protocol describes a standardized surgical method for the elastase-induced AAA model through the direct application of elastase to the adventitia of infrarenal abdominal aorta in mice.

עכבר בטן מפרצת אבי העורקים מודל המושרה על ידי יישום פרי וסקולרי של אלסטאז

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. ...

A Calcium Phosphate-Induced Mouse Abdominal Aortic Aneurysm Model

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that occurs worldwide and is characterized by irreversible dilation of the abdominal aorta. Currently, several chemically induced murine AAA models are used, each simulating a different aspect of the pathogenesis of AAA. The calcium phosphate-induced AAA model is a rapid and cost-effective model compared to the angiotensin II- and elastase-induced AAA models. The application of CaPO4 crystals to the mouse aorta results in elastic fiber degradation, loss of smooth muscle cells, inflammation, and calcium deposition associated with aortic dilation. This article introduces a standard protocol for the CaPO4-induced AAA model. The protocol includes material preparation, the surgical application of the CaPO4&#160;to the adventitia of the infrarenal abdominal aorta, the harvesting of aortas to visualize aortic aneurysms, and histological analyses in mice.

This protocol describes a calcium phosphate-induced abdominal aortic aneurysm (AAA) mouse model to study the pathological features and molecular mechanisms of AAAs.

מודל מפרצת אבי העורקים בבטן הנגרמת על-ידי סידן פוספט על-ידי עכבר

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that occurs worldwide and is characterized by irreversible dilation of the ...